Recognition of microbes by Toll-like receptors (TLRs) triggers the induction of proinflammatory cytokines and other immune mediators, which serves as an important innate immune mechanism against infections. However, uncontrolled secretion of inflammatory mediators causes chronic inflammatory diseases or even a devastating acute illness, septic shock. The long-range goal of this project is to dissect the signaling pathways involved in TLR-mediated induction of inflammatory mediators. This knowledge is important for rational design of more effective anti-inflammatory therapies. Over the past few years, we have made seminal findings demonstrating a pivotal TLR signaling axis: the IKK/Tpl2 axis. IKK is known as a kinase that activates the transcription factor NF-(B by mediating phosphorylation and degradation of the NF-(B inhibitor I(B, whereas Tpl2 is a MAP3K that activates the MAP kinase ERK through phosphorylating the ERK kinase, MEK1. Interestingly, Tpl2 is sequestered by an I(B-like molecule, p105, and the activation of Tpl2 requires IKK-mediated p105 phopshorylation and degradation. Thus, the IKK/Tpl2 signaling axis controls two major TLR pathways, the NF-(B and ERK pathways. The overall objective of this continuation application is to understand how IKK regulates Tpl2 and how the IKK/Tpl2 signaling axis is activated by upstream TLR signals. The proposed project is based on strong preliminary data from our laboratory. In particular, our studies have revealed the involvement of IKK/Tpl2 functional interplay and novel signaling factors in TLR-stimulated p105 degradation and Tpl2 activation. We have further shown that in addition to mediating Tpl2 activation, IKK controls the fate of activated Tpl2, a function that may prevent prolonged Tpl2 activation. Additionally, we made significant progress toward understanding the mechanism that connects the IKK/Tpl2 axis to upstream TLR signals. Our genetic evidence suggest that a recently identified E3 ubiquitin ligase, Pellino1, mediates IKK/Tpl2 activation by specific TLRs. Together, these innovative preliminary results form a solid foundation for the studies proposed in this application. We will perform three specific aims to accomplish our overall objective. (1) Examine the molecular mechanism of IKK- dependent Tpl2 activation. (2) Examine the role of IKK in regulating the fate of Tpl2. (3) Examine how the IKK/Tpl2 signaling axis is regulated by upstream TLR signals